Overexpression of the epidermal growth factor receptor (EGFR) and its association with the tyrosine kinase, c-Src, is correlated with increased cellular proliferation and tumorigenesis. Previous studies have shown that EGFR and c-Src co-overexpression and association leads to the c-Src-mediated phosphorylation of tyrosine 845 of the EGFR and that mutation of Tyr(845) ablates epidermal growth factor (EGF)-induced DNA synthesis. Here, we investigate the contribution of the signal transducers and activators of transcription (STAT5b) in the signaling pathways regulated by EGFR and c-Src overexpression in human breast tumor cell lines as well as in a mouse fibroblast model (C3H10T1/2). We demonstrate that 1) activation of STAT5b by EGF requires overexpression of the EGFR, 2) co-overexpression of c-Src alone does not result in EGF-induced activation of STAT5b but enhances that seen in EGFR-overexpressing cells, and 3) EGF-induced tyrosine phosphorylation of STAT5b requires Tyr(845) of the EGFR. Furthermore, the stable overexpression of a kinase-defective c-Src in the context of EGFR overexpression results in a decrease in the tyrosine phosphorylation of STAT5b in response to EGF and a more dramatic decrease in EGF-induced transcriptional activation of STAT5b, suggesting an integral role for c-Src in the physiological actions of STAT5b. Using a dominant negative STAT5b, we provide evidence that one such physiological action is to mediate EGF-induced DNA-synthesis. Finally, the use of site-specific tyrosine mutants demonstrates that EGF-induced phosphorylation of STAT5b involves not only tyrosine 699 of STAT5b, which is required for its transcriptional activation, but also three previously identified tyrosines in the C terminus of STAT5b (Tyr(725)/Tyr(740)/Tyr(743)).

We have investigated and compared GH and epidermal growth factor (EGF) signaling in primary human skin fibroblasts from normal subjects and subjects with GH-binding protein-positive Laron syndrome (LS). In normal human fibroblasts, GH and EGF activate the tyrosine phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT5b; in LS fibroblasts, EGF does, but GH does not. GH also activates the tyrosine phosphorylation of Janus kinase (JAK)2 in normal, but not LS, fibroblasts. Similarly, both GH and EGF activate MAPK in normal fibroblasts, but only EGF does in the LS fibroblasts. As in the 3T3-F442A mouse preadipocyte cell line, GH signaling to mitogen-activated protein kinase is partially inhibited by wortmannin treatment, indicating a role for phosphatidylinositol 3-kinase (PI3K) in this signaling pathway. The exogenous expression of the GH receptor in one family of LS fibroblasts (H1) but not the other (M) restores signaling to a STAT5 reporter element. Together, these results indicate that the mechanism of defective GH signaling in two families of LS fibroblasts are different but that both occur at a level close to, and specific for, the GH receptor.